Agnew B J, Duman J G, Watson C L, Coling D E, Forte J G
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
J Cell Sci. 1999 Aug;112 ( Pt 16):2639-46. doi: 10.1242/jcs.112.16.2639.
Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H, K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H, K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H, K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.
培养的兔壁细胞用于评估对盐酸分泌激活剂和抑制剂的形态学反应。免疫荧光用于定位质子泵蛋白H、K-ATP酶以及顶端膜-细胞骨架连接蛋白埃兹蛋白；荧光标记的鬼笔环肽用作F-肌动蛋白的标志物。用促分泌剂处理健康对照壁细胞导致顶端膜泡过度肿胀，推测是由于盐酸和水的积累。因此，在分泌激活级联反应的各个步骤起作用的抑制剂可阻断与刺激相关的顶端膜泡肿胀。当分泌被阻止富含H、K-ATP酶的微管泡向顶端膜泡转运的药物（如H2受体拮抗剂和蛋白激酶A抑制剂）阻断时，可维持一般的静息形态。ME-3407（渥曼青霉素的功能类似物）在阻止H、K-ATP酶重新分布以及使埃兹蛋白从顶端膜泡中脱离定位方面具有独特性。当分泌被抑制H+泵或诱导H+回流的药物阻断时，H、K-ATP酶向顶端膜泡的转运发生，但与盐酸和水积累相关的大泡肿胀大大减轻。这些数据支持盐酸分泌的膜循环/募集假说，即富含H、K-ATP酶的微管泡从细胞质区域募集到顶端表面，并且它们与提出无论微管泡形状如何都与顶端质膜相邻的模型不一致。这些研究还证明了壁细胞培养模型在区分各种抑制剂和抗分泌剂的一般作用位点方面的实用性。
